Difficidin and derivative antibacterials

ABSTRACT

Difficidin and derivative antibacterial compounds of the formula: &lt;IMAGE&gt; (I.)   where Ra and Rb are members independently selected from the group consisting of hydrogen; alkali metal and alkaline earth metal cations; ammonium; and substituted ammonium; and R1 is hydrogen or hydroxy.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel antibacterial compound referredto herein generally as "difficidin", and derivative compounds, and to aprocess for preparing, isolating, and purifying said compounds. Asemployed herein, the coined term "difficidin" refers to the compound ofFormula I below wherein R¹ is hydrogen, and R_(a) and R_(b) have therecited meanings, thus affording various salt forms thereof.Correspondingly, the term "oxydifficidin", as used herein, refers to thecompound of Formula I below wherein R¹ is hydroxy, and R_(a) and R_(b)have the indicated meanings. The novel antibacterial compounds areobtained by microbiological cultivation of Bacillus subtilis, MB 3575,and MB 4488 deposited with the American Type Culture Collection,Rockville, Md. under the designations ATCC 39374 and 39320,respectively.

The novel antibacterial compounds of the present invention, difficidinand derivative compounds, are broad spectrum antibacterials with goodpotency against aerobic microorganisms and exceptional potency againstanaerobic microorganisms. They also exhibit good in vitro activityagainst microorganisms that have developed resistance to conventionalantibacterials. They may be used parenterally in the treatment of grampositive and gram negative bacterial infections.

2. Brief Description of the Prior Art

Proticin is a known antibacterial compound which has been described as aphosphorous-containing, strongly unsaturated amorphous compound withbroad activity spectrum, especially against gram negative pathogens. Itis said to have been produced by fermentation of a strain identified asa form of Bacillus licheniformis. A more detailed description of itspreparation, characteristics, and antibacterial activity may be found inthe following references: (1) Prave, P. et al., J. Antibiotics 25 (1):1-3, 1972; (2) Vertesy, L., J. Antibiot. 25 (1): 4-10, 1972; (3)Nesemann, G., et al., Naturwissenschaften 59 (2): 81-82, 1972; (4) Ger.Offen. No. 2,035,812 (Vertesy. L. et al.), "Proticin from Bacilluslicheniformis", Farbwerke Hoechst A.-G., 18 July 1970; and (5) Brit.1,350,271, "Proticin Production by Bacillus licheniformis fermentation,"Farbwerke Hoechst A.-G., Apr. 18, 1974. (ref. Chem. Abstracts 81:48531y, p 30b, 1974).

However, despite the repeated reference to the specific antibioticcompound proticin, the above references fail to characterize any singlecompound, and the spectral data provided therein is consistent with anumber of possible compounds. Sine Vertesy Reference (2) above describesthe presence of a methyl group at only one of the double bonds, whereasthe compounds of the present invention have two such methyl groups, ithas been concluded that the novel antibacterial compound of the presentinvention, wherein R¹ is hydroxy (oxydifficidin) is chemically distinctfrom the so-called proticin, described in the above references. On theother hand, the novel antibacterial compound of the present inventionwherein R¹ is hydrogen (difficidin) is readily distinguished from thecompound(s) of the references since it has a different molecular weight.

Chemically, difficidin is a 22-membered macrocyclic polyene lactonephosphate ester with asymmetric centers at C4, C5, C15 and C21, asillustrated by Formula I.

As can be seen, there are two chief compounds within Formula I. Onecompound is that wherein R¹ is hydrogen, which as already mentioned,shall be referred to hereinafter as "difficidin"; and the other compoundis that wherein R¹ is hydroxy, which shall be referred to hereinafter as"oxydifficidin".

Biologically, difficidin has been found to be more potent thanoxydifficidin against aerobic microorganisms such as Pseudomonasaeruginosa and Staphyloccoccus aureus. In most cases it was found to befour times more effective against S. aureus, Ps. aeruginosa, Salmonellatyphimurium, Proteus, and Serratia marcescens than oxydifficidin.Difficidin and oxydifficidin are also active against a wide spectrum ofanaerobic bacteria.

DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS

In accordance with the present invention there is provided the novelantibacterial compound, difficidin, and its derivatives of the formula:##STR2## where R_(a) and R_(b) are members independently selected fromthe group consisting of hydrogen; alkali metal and alkaline earth metalcations; ammonium; and substituted ammonium; and R¹ is hydrogen orhydroxy.

The ammonium cation may be substituted in known manner, preferably withlower alkyl, for example.

Difficidin has been prepared and utilized as the potassium phosphatesalt, being a changing equilibrium of the mono- and di-potassium salts.This salt form has been employed due to its ease of preparation and thepossible instability of the acid form, where R_(a) =R_(b) =H, whichexists at a low pH.

Difficidin and its derivatives may be prepared by microbiologicalcultivation of Bacillus subtilis, MB 3575 and MB 4488 deposited with theAmerican Type Culture Collection, Rockville, Md., from which it isavailable without restriction under the accession numbers ATCC 39374 and39320, respectively.

The bacillus, or its variants and mutants may be cultivated inaccordance with well known microbiological processes, either on agarslant tubes, or under submerged conditions in Erlenmeyer flasks orfermentors, utilizing nutrient media or nutrient solutions generallyemployed for cultivating microorganisms.

In the present invention, difficidin and its derivatives are producedduring cultivation of the microorganism, for example, Bacillus subtilisATCC 39320 at a temperature of about 28° C., under aerobic condition.The composition of the nutrient medium may be varied over a wide range.The essential assimilable nutrient ingredients are; a carbon source, anitrogen source, a source of inorganic elements including phosphorus,sulfur, magnesium, potassium, calcium and chlorine. Cultivation is mostproductive under neutral pH conditions, preferably from about 6.0 to7.0.

Typical sources of carbon include, glucose, dextrin, starches, glyceroland the like. Typical nitrogen sources include vegetable meals (soy,cottonseed, corn, etc.), meat flours or animal peptones, distillerssolubles, casamino acids, yeast cells, various hydrolysates (casein,yeast, soybean, etc.), yeast nucleic acids and amino acids.

Mineral salts such as the chlorides, nitrates, sulfates, carbonates andphosphates of sodium, potassium, ammonium, magnesium and calcium providea source of essential inorganic elements. The nutritive medium may alsocontain a number of trace elements such as iron, copper, manganese, zincand cobalt.

If excessive foaming is encountered during the cultivation, antifoamingagents such as vegetable oils, lard oil and polypropylene glycol may beadded to the fermentation medium prior to, or during the course of thefermentation. The maximum yield of difficidin can be achieved withinfrom about 20 to 120 hours, and is culture dependent. The inoculum forthe fermentation can be provided from suspensions, slants, frozen cellsor freeze-dried preparations.

In addition to the conventional cultivation processes described above,there may also be employed continuous processes, such as that describedin Methods in Microbiology, Vol. 2, Academic Press, London--New York,1970, pp. 259-328. In such systems the bacillus can be maintained forextended periods of time in a steady state without spontaneous mutationsor other degenerations becoming evident.

It is to be understood that for the production of difficidin and itsderivatives, the present invention is not limited to the use of Bacillussubtilis ATCC 39374 or 39320. It is especially desired and intended toinclude the use of natural or artificial mutants produced from thedescribed organisms, or other variants of Bacillus subtilis ATCC 39374or 39320 as far as they can produce difficidin and its derivatives. Theartificial production of mutant Bacillus subtilis may be achieved by aconventional operation such as X-ray or ultraviolet (UV) radiation, orby the use of chemical mutagens such as; nitrogen mustards,nitrosoguanidine, camphor and the like, or by means of recombinant DNAtechnology.

MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERISTICS OF BACILLUS SUBTILISATCC 39320

The morphological and physiological properties of ATCC 39320 are asfollows:

Morphology: gram positive, non-vacuolated vegetative rods with roundedends; average size 0.9×2.3-3.6μ; occurring singly. Rods are motile.Spores are produced under aerobic conditions. Spores are 0.5×1.0μ(average size), oval to cylindrical, predominantly central, sporangianot swollen.

Colonial appearance: flat, round with irregular edge, surface dull, edgebecoming opaque as colony ages. Dull, wrinkled entire pellicle onsurface of broth. No pigmentation on trypticase soy agar. Growth at 28°C., 37° C., no growth at 60° C.

Positive reactions: Catalase, Voges-Proskauer, gelatin, nitratereduction, utilization of citrate, acid from glucose, arabinose,mannitol, xylose, sorbitol and sucrose, hydrolysis of starch.

Negative reactions: urease, indole, utilization of propionate, argininedihydrolase, acid from rhamnose and mellibiose, no growth in anaerobicagar (stabs or plates incubated in anaerobic jars), no growth in glucosebroth or nitrate broth under anaerobic conditions.

Comparison with culture descriptions in Bergey's Manual of DeterminativeBacteriology, Eighth Edition, Williams & Wilkins, 1974, and Gordon, R.E., Haynes, W. C. and Pang, C. H. (1973), The Genus Bacillus,Agriculture Monograph No. 427, U.S. Department of Agriculture,Washington, D.C., indicate that MB 4488/ATCC 39320 is a strain of knownspecies Bacillus subtilis.

MORPHOLOGICAL AND PHYSIOLOGICAL CHARACTERISTICS OF BACILLUS SUBTILISATCC 39374

The morphological and physiological properties of ATCC 39374 are thesame as those indicated above for ATCC 39320, except with respect to theappearance of the colonies of the microorganism, which are as follows:

Colonial appearance: At 24 hours, raised, round, mucoid. As colony ages,edge becomes dry, opaque and irregular. Central mucoid area continues todry, becoming opaque and wrinkled.

Dull, wrinkled entire pellicle on surface of broth. No pigmentation ontrypticase soy agar. Growth at 28° C., 37° C., no growth at 60° C.

PRODUCTION OF DIFFICIDIN AND ITS DERIVATIVES

A. A process for preparing the antibacterial compound difficidin and itsderivatives, or one of their salts, but in which oxydifficidin isproduced in greater proportion, involves the cultivation ofmicroorganisms which form difficidin and its derivatives and belong tothe strain of Bacillus subtilis ATCC 39320 at a temperature ranging from20° to 40° C. for from 24 to 120 hours by means of an aqueous nutrientsolution which contains a source of carbon, a source of nitrogen,nutrient salts and trace elements, until the nutrient solution containsconsiderable amounts of difficidin and its derivatives, after which thedifficidin and its derivatives are isolated from the culture andconverted, if desired, into a salt with a pharmaceutically acceptablebase.

During cultivation the pH value of the nutrient medium changes fromneutral to slightly acidic; in general the addition of a buffer solutionis not necessary, although one could be employed as a precaution. Thecultivation is suitably stopped after 2 to 4 days, since a favorableyield is obtained after this period; the nutrient solution then containsa substantial amount of oxydifficidin. It has been found that the ATCC39320 culture produces a preponderance of oxydifficidin, as compared todifficidin, but that the overall titer is lower than with the ATCC 39374culture described below.

B. Another, and preferred, process for preparing the antibacterialcompound difficidin and its derivatives, or one of their salts with apharmaceutically acceptable base, but in which difficidin is produced ingreater proportion, involves the cultivation of microorganisms whichform difficidin and its derivatives and belong to the strain of Bacillussubtilis ATCC 39374 at a temperature ranging from 20° to 40° C. for from24 to 120 hours by means of an aqueous nutrient solution which containsa source of carbon, a source of nitrogen, nutrient salts and traceelements, including particularly cobalt, until the nutrient solutioncontains considerable amounts of difficidin and its derivatives afterwhich the difficidin and its derivatives are isolated from the cultureand converted, if desired, into a salt with a pharmaceuticallyacceptable base.

As already mentioned, the process utilizing the strain of Bacillussubtilis ATCC 39374 is also preferred because it has been found toproduce greater proportions of difficidin than oxydifficidin and highertiters than the Bacillus subtilis ATCC 39320 microorganism utilized inthe process described under "A" above.

It has been found that the addition of cobalt, in amounts of for example0.1 g/l, causes preferential production of difficidin by themicroorganism, to the extent of as much as a 3:1 weight ratio to theamount of oxydifficidin produced. Various water soluble salts of cobaltmay be employed, for example cobalt chloride hexahydrate, which ispreferred.

Similarly, it has been found that the addition of phosphate, in amountsof for example 100 p.p.m., in the presence of cobalt, causespreferential production of difficidin by the Bacillus subtilis culture,as compared to the production of oxydifficidin. On the other hand,preferential production of oxydifficidin can be accomplished by additionof manganese, for example, manganese chloride tetrahydrate, in amountsof, for example, 0.025 g/l. However, these findings are preliminary innature and are included only as an indication of potential advantageousmanipulation of the culture medium for the purpose of affectingproduction of either difficidin or oxydifficidin, as may be preferred.

ISOLATION OF DIFFICIDIN AND ITS DERIVATIVES

In order to isolate difficidin and its derivatives, the culture of thebacillus may first be clarified by centrifugation, which, however,results in a considerable proportion of difficidin and its derivativesremaining in the cell mass. This crude product may be purified bychromatography using an appropriate adsorbent such as polymeric organicbased resins, such as XAD-2 and HP-20 resins, and polymeric silicic acidbased resins, such as LiChroprep RP-18 resin and Watman ODS resins, bothof which are silica with a chemically bonded outer layer of C₁₈ H₃₇residues. Difficidin and its derivatives are eluted from the adsorbentwith suitable polar solvents or mixtures of solvents, after whichaqueous extraction of difficidin and its derivatives is carried out. Thedifficidin and oxydifficidin are then separated from each other andstructurally related components by HPLC.

ANTIBACTERIAL ACTIVITY

Using standard in vitro assay techniques, minimal inhibitoryconcentrations (MIC) have been determined for difficidin andoxydifficidin against a number of bacteria. The results obtained areillustrated in Table I below.

                  TABLE I                                                         ______________________________________                                                        MIC (μg/ml) Range                                                            Oxy-                                                        Organism (10.sup.6 CFU)                                                                         difficidin                                                                              Difficidin                                        ______________________________________                                        Staphylococcus aureus (3)*                                                                       32-128    8-16                                             Streptococcus faecalis (1)                                                                      128       128                                               Escherichia coli (4)                                                                             4-16     8                                                 Salmonella typhimurium (1)                                                                      32        8                                                 Enterobacter (3)  32-64      8-16                                             Klebsiella pneumoniae (2)                                                                        8        8                                                 Proteus (3)        2-32     0.5-8                                             Pseudomonas aeruginosa (3)                                                                      16-32     8                                                 Serratia marcescens (1)                                                                         16        4                                                 ______________________________________                                         *number of strains tested.                                               

Further evaluations of the antibacterial spectrum of difficidin andoxydifficidin were undertaken, especially as compared to theantibacterial spectrum of the valuable antibiotic cefoxitin, which iswell known for its wide spectrum of antibacterial activity. An in vitroagar dilution assay was used, inoculum 10⁶ CFU per spot. Table II belowillustrates the results obtained for various aerobic bacteria, and TableIII illustrates the results obtained for various anaerobic bacteria.

                  TABLE II                                                        ______________________________________                                                    MIC (μg/ml)                                                                  Oxy-       Dif-                                                 Aerobic Bacteria                                                                            difficidin ficidin   Cefoxitin                                  ______________________________________                                        S. aureus Gm.sup.R Meth.sup.R                                                               32.0       8.0       >128                                       S. aureus     >128.0     16.0      2                                          S. aureus     32.0       8.0       2                                          Strep. faecalis                                                                             >128.0     >128.0    >128                                       E. coli TEM 2.sup.+                                                                         16.0       8.0       4                                          E. coli TEM 2.sup.+ DC 2                                                                     8.0       8.0       1                                          E. coli DC 2   4.0       8.0       1                                          E. coli        4.0       8.0       >128                                       Sal. typhimurium                                                                            32.0       8.0       8                                          Ent. cloacae P99.sup.+                                                                      32.0       16.0      >128                                       Ent. cloacae P99.sup.-                                                                      64.0       8.0       16                                         Ent. aerogenes                                                                              32.0       8.0       >128                                       K. pneumoniae Kl.sup.+                                                                       8.0       8.0       4                                          Prot. vulgaris                                                                               8.0       0.5       16                                         Prot. morganii Sm.sup.R                                                                      2.0       2.0       16                                         Prot. mirabilis Gm.sup.R                                                                    32.0       8.0       32                                         Ps. aeruginosa RPL 11.sup.+                                                                 16.0       8.0       >128                                       Ps. aeruginosa                                                                              32.0       8.0       >128                                       Ps. aeruginosa                                                                              32.0       8.0       >128                                       Ser. marcescens                                                                             16.0       4.0       64                                         ______________________________________                                    

                  TABLE III                                                       ______________________________________                                                       MIC (μg/ml)                                                                  Oxy-      Dif-      Ce-                                      Anaerobic Bacteria                                                                             difficidin                                                                              ficidin   foxitin                                  ______________________________________                                        Actinomyces naeslundii                                                                         64        >128      0.015                                    Eubacterium limosum                                                                            >128      >128      0.25                                     Propionibacterium acnes                                                                        8         1         ≦0.008                            Peptostreptococcus                                                                             0.06      0.06      ≦0.008                            anaerobius                                                                    Clostridium perfringens                                                                        >128      >128      0.5                                      NCCLS control strain                                                          C. ramosum       2         2         4                                        C. difficile Clind.sup.r,                                                                      0.06      0.125     64                                       Fox.sup.r                                                                     C. difficile Fox.sup.r                                                                         0.06      0.06      128                                      Bifidobacterium dentium                                                                        >128      32        4                                        Fox.sup.r                                                                     Bacteriodes fragilis                                                                           0.25      0.125     2                                        Metr.sup.r, Pen.sup.r                                                         B. fragilis Pen.sup.r                                                                          0.125     0.125     8                                        B. fragilis Clind.sup.r Fox.sup.r Tet.sup.r                                                    0.25      0.25      32                                       B. fragilis      0.25      0.06      4                                        NCCLS control strain                                                          B. distasonis Pen.sup.r Fox.sup.r Clind.sup.r                                                  0.06      0.06      16                                       B. distasonis Pen.sup.r Fox.sup.r                                                              >128      >128      64                                       B. ovatus Pen.sup.r                                                                            0.25      0.125     8                                        B. thetaiotaomicron Clind.sup.r                                                                0.25      0.25      16                                       B. thetaiotaomicron Clind.sup.r,                                                               0.25      0.06      16                                       NCCLS control strain                                                          Fusobacterium mortiferum Fox.sup.r                                                             0.125     0.03      16                                       Veillonella alcalescens                                                                        0.06      0.25      0.5                                      ______________________________________                                    

The antibacterial efficacy of difficidin and its derivatives was furtherdetermined in standard mouse protection tests. An experimental systemicinfection was produced in CD1 female mice (Charles River BreedingLaboratories, Wilmington, MA) by the intraperitoneal injection of asuitably diluted broth culture of Klebsiella pneumoniae MB 4005. Thetest antibacterial compounds and the control drug (streptomycin) wereadministered parenterally (intraperitoneally) immediately afterinfection and again 6 hours later. At least 4 fourfold dilutions of eachagent were tested. There were 5 mice at each dose level. All mice wereobserved for a period of seven days after which the median effectivedose (ED₅₀) was calculated by the method of Knudsen and Curtis (Knudsen,L. F. and J. M. Curtis. J. Amer. Statist. Assn. 42: 282-296, 1947).Under the conditions of the test, all infected untreated mice diedwithin 48 hours.

Mice were protected by the administration of the test antibacterialcompounds or streptomycin at the median effective doses shown in TableIV below.

                  TABLE IV                                                        ______________________________________                                                            ED.sub.50      MIC                                        Test No.                                                                              Antibiotic  mg/kg × 2 i.p. doses                                                                   μg/ml                                   ______________________________________                                        1       Oxydifficidin                                                                             12.25          8                                                  Streptomycin                                                                              0.38                                                      2       Oxydifficidin                                                                             14.85                                                             Streptomycin                                                                              1.41                                                      3       Difficidin  1.56           8                                                  Streptomycin                                                                              0.38                                                      ______________________________________                                    

These data show that all test antibacterial compounds were effective inprotecting mice from an otherwise lethal infection with Klebsiellapneumoniae.

From the foregoing in vitro and in vivo data it is expected that aneffective antibacterial amount of difficidin and its derivatives wouldbe on the order of 5 mg/kg to 20 mg/kg in mammals. Difficidin and itsderivatives are effective for treatment of gram negative and grampositive infections as described above, and may be administeredintravenously, intramuscularly, or subcutaneously, either alone or incombination with a pharmaceutical carrier. The ultimate choice of routeand dose should be made by an attending physician and based upon thepatient's unique condition.

Combinations of difficidin and its derivatives with appropriatepharmaceutical carriers are accomplished by methods well known to thepharmacist's art. For purposes of subcutaneous (s.c.) administration,solutions of difficidin and its derivatives are generally employed, forexample, sterile aqueous or alcoholic solutions. Such solutions shouldbe suitably buffered if necessary and the liquid diluent may first berendered isotonic with saline or glucose. These aqueous and alcoholicsolutions are also suitable for intravenous (i.v.) injections.

The following examples illustrate the preparation and isolation ofdifficidin and oxydifficidin from Bacillus subtilis ATCC 39320 and ATCC39374.

EXAMPLE 1 Production of Difficidin and Oxydifficidin Using ATCC 39320

    ______________________________________                                        1.  "A" Stage  Culture MB 4488, lyophilization tubes, is                                     maintained in the lyophilized state in a 1.0                                  ml ampule containing 0.15 ml of a skim milk                                   suspension of the culture.                                     2.  "B" Stage                                                                     Vessel:    250 ml 3-baffled Erlenmeyer flask                                             containing 50 ml medium per flask.                                                  Wt/Vol                                                   Medium:    Dextrose        1       g/l                                                   Soluble Starch  10      g/l                                                   Beef Extract    3       g/l                                                   Ardamine pH     5       g/l                                                   NZ-Amine Type E 5       g/l                                                   MgSO.sub.4 7 H.sub.2 O                                                                        0.05    g/l                                                   1.34 M Phosphate Buffer                                                                       0.02%   Vol/vol                                               CaCO.sub.3 (After pH                                                                          0.05%                                                         Adjustment to 7.0-7.2)                                                        Phosphate Buffer                                                              KH.sub.2 PO.sub.4                                                                             91      g/l                                                   Na.sub.2 HPO.sub.4                                                                            95      g/l                                            Inoculum:  Contents of one Lyo tube into each                                            "B" flask.                                                         Incubation:                                                                              24 hours at 28° C. on a rotary                                         shaker with a 2" throw rotating at                                            220 RPM.                                                           Sterility: Streak plates and gram stain.                                  3.  "C" Stage                                                                     Vessel:    2 liter 3-baffled Erlenmeyer flask                                            containing 500 ml medium.                                          Medium:    Same as "B" stage.                                                 Inoculum:  10 ml from "B" stage.                                              Incubation:                                                                              Same as "B" stage.                                                 Sterility: Same as "B" stage.                                             4.  "E" Stage                                                                     Vessel:    750 liter stainless steel                                                     fermentor.                                                     Medium:    Dextrin         40      g/l                                                   Solulac         7       g/l                                                   Ardamine YEP    5       g/l                                                   Cobalt Chloride 50      mg/l                                                  Polyglycol 2000 1       ml/l                                                  Pre-sterile pH  7.3                                                Sterilization:                                                                           20 minutes at 121° C.                                       Inoculum:  1 liter "C" stage.                                                 Fermentor  501 liters.                                                        Volume A.I.:                                                                  Temperature:                                                                             28° C.                                                      Airflow:   5 CFM (30 hrs) then 6 CFM.                                         Agitation: 130 RPM (12 hrs) then 110 RPM.                                     Pressure:  13 PSI.                                                            Defoamer:  Polyglycol 2000                                                    Cycle Time:                                                                              92 hours.                                                          Sterility: Microscopic examination and streak                                            plates at 28° and 37° C.                              Physiology:                                                                                                        Difficidin                              Age             % Cell                Titer                                   (hrs)  pH       Volume   NH.sub.3                                                                              PO.sub.4                                                                           (μg/ml)                              ______________________________________                                         0     6.5      4        55      380                                           9     6.0      5        40      200                                          21     --       14       80      210                                          33     6.4      --       105     220                                          45     6.4      --       70      250  10.8                                    57     6.3      --       15      180  7.9                                     69     6.4      --       10      210  --                                      81     6.0      4        25      250  7.4                                     93     6.0      2        65      275  6.6                                     ______________________________________                                    

EXAMPLE 2 Isolation and Purification of Difficidin and Oxydifficidin A.Preliminary isolation of antibacterial complex

Approximately 420 L of whole broth (pH 5.9) from the fermentation batchdescribed in Example 1 was harvested at 92 hours. The broth wasclarified using a Sharples centrifuge. The 416 L of clarified broth wasadjusted to pH 5.0 with 500 ml of 4N hydrochloric acid and then adsorbedonto 38 L of Rohm and Haas Amberlite XAD-2 resin at 4 L/min. The resinwas washed at 4 L/min. with 120 L of cold, distilled deionized water.The antibacterial complex was eluted from the resin with 160 L of ethylacetate at 4 L/min. The lower aqueous layer in the eluate was removedand discarded. The eluate was dried by agitation for 30 minutes overanhydrous sodium sulfate. The drying agent was removed by filtration andthe filtrate was concentrated under vacuum to 2.65 L. A 1 liter portionof water was added to the concentrate and under gentle stirring the pHof the lower aqueous phase was adjusted to 8.5 with 130 ml of 2.5Nsodium hydroxide. The mixture was stirred vigorously for 2 minutes. Thelayers were allowed to separate. The lower aqueous extract was decantedand the upper organic layer was extracted twice more, each time with 500ml of water and sufficient 2.5N sodium hydroxide to bring the pH to 8.5during extraction. The three aqueous extracts were combined and adjustedto pH 6.9. The extract volume was 2.4 L. The 2.1 L spent ethyl acetatesolution contained 94 g of total solids.

Difficidin and oxydifficidin were present to various extents in both theaqueous extract and the spent ethyl acetate fraction. Step B belowdescribes the isolation of difficidin and oxydifficidin from the spentethyl acetate. Step C below describes the isolation of difficidin fromthe aqueous extract. Step D describes an alternative isolation procedureand corresponding analytical evaluation.

B. Isolation of Difficidin and Oxydifficidin

A 0.4 ml sample of 2.1 L spent ethyl acetate fraction from Step A abovewas evaporated to an oily residue and taken up in 0.7 ml of 7:3methanol-0.01M potassium phosphate pH 7.0. A 0.4 ml aliquot of thesolution was chromatographed in the same solvent at 28° C. on a DupontZorbax ODS column (4.6 mn ID×25 cm), 10 micron. The flow rate was 1ml/min. and column effluent was monitored at 275 nm using a LaboratoryData Control Spectra Monitor I ultraviolet detector equipped with a 10mm pathlength flow cell and a Honeywell Electronik 195 recorder. Eluatewas collected in 0.5 ml fractions and assayed by agar disc diffusionagainst Vibrio percolans MB 1272. Table V summaries bioassay results.

The data in Table V show the presence of two distinct antibacterialsubstances in spent ethyl acetate. The activity in Fractions 11 and 12is due to the antibacterial oxydifficidin. The activity in Fractions61-65 is due to the antibacterial difficidin.

                  TABLE V                                                         ______________________________________                                        In vitro Vibrio percolans                                                     Bioassay Results of Column Fractions                                                  Zone     Peak Assign.                                                         Diameter and Elution     Component                                    Fraction                                                                              (mm)*    Time            Assignment                                   ______________________________________                                        1-10     0       Peak 1, 336 sec Oxydifficidin                                11      23                                                                    12      19                                                                    61      12       Peak 2, 1844 sec                                                                              Difficidin                                   62      12                       Component                                    63      12                                                                    64      12                                                                    65      12                                                                    66-120   0                                                                    ______________________________________                                         *agar disc diffusion, 40 μl on 3/8" disc                              

C. Isolation of Antibacterial Difficidin--Method 1

The 2.4 L of combined aqueous extract (66 g total solids) from Step Aabove was concentrated to 2.1 L to remove most of the dissolved ethylacetate. The concentrate was diluted with 0.22 L of 1M potassiumphosphate pH 7.0 and adsorbed at 50 ml/min. on 1 liter of MitsubishiDiaion HP-20 resin. The resin was washed successively with 3 L ofdistilled water, 3 L of 1:1 methanol-0.1M potassium phosphate pH 7.0, 3L of 65:35 methanol-phosphate buffer, and 3 L of 9:1 methanol-distilledwater. The antibacterial complex was eluted at 50 ml/min. with 3.9 L ofmethanol, collected as a 1.45 L forerun fraction and a 2.45 L rich cut.The forerun was enriched in oxydifficidin while the rich cut wasenriched in difficidin. The rich cut (0.55 g total solids) wasconcentrated to 50 ml and diluted with 116 ml of 0.1M potassiumphosphate pH 7.0. The solution was charged on a 74 ml column of E. MerckLiChroprep RP-18, 25-40 micron resin at 5 ml/min. The column wassuccessively developed with methanol-water mixtures of increasingmethanol concentration (Table VI). The flow rate was 5 ml/min. andfractions were 7.5 ml each.

                  TABLE VI                                                        ______________________________________                                        Chromatography of Crude Difficidin Component                                  on LiChroprep RP-18                                                           Fraction   Solvent        Volume                                              ______________________________________                                         1-27      1:1 methanol-water                                                                           7.5 ml/fraction                                      28-60     55:45 methanol-water                                                                         7.5 ml/fraction                                      61-95     60:40 methanol-water                                                                         7.5 ml/fraction                                      96-130    65:35 methanol-water                                                                         7.5 ml/fraction                                     131-190    70:30 methanol-water                                                                         7.5 ml/fraction                                     ______________________________________                                    

Based on HPLC analysis and bioassay of fractions, an antibacterialeluted in Fractions 109-121 which exhibited the same HPLC retention timeas Peak 2 (difficidin component) of Table V. Fractions 109-121 werecombined as crude difficidin component rich cut.

The rich cut was concentrated to 8.2 ml and diluted with 0.9 ml of 1Msodium citrate pH 5.4 and 6.1 ml of methanol. The solution was adsorbedat 2 ml/min. onto a 17 ml column of E. Merck LiChroprep RP-18 resin,25-40 micron in 4:6 methanol-0.1M sodium citrate pH 5.5. The column waswashed at the same flow rate with 30 ml of 4:6 methanol-citrate buffer.The column was then developed with 75:25 methanol-citrate (Fractions1-35) and 8:2 methanol-citrate (Fractions 36-70). All fractions were 2ml. Based on TLC and UV analysis, Fractions 42-65 were combined andconcentrated to 6 ml. The resulting white suspension was diluted with 5ml of 0.1M potassium phosphate pH 7.2 and sufficient methanol (7 ml) toredissolve the insoluble material. The solution was desalted in thefollowing manner. The solution was adsorbed on 8 ml of LiChroprep RP-18resin in 3:7 methanol-0.1M potassium phosphate pH 7.0. The resin waswashed successively with 24 ml of 3:7 methanol-phosphate buffer and 24ml of 3:7 methanol-distilled water. The difficidin component was elutedwith 30 ml of 9:1 methanol-distilled water. The eluate was analyzed byHPLC using a Dupont Zorbax ODS column (4.6 mm ID×25 cm) at ambienttemperature in 81:19 methanol-0.1M sodium citrate pH 5.5. The flow ratewas 2 ml/min. Approximately 94% of the UV absorbance of the eluate at275 nm was attributable to difficidin component (retention time 379sec.). An impurity with retention time 418 seconds accounted for theremaining 6% of 275 nm absorbance. The impurity was removed by carefulchromatography on LiChroprep RP-18 resin. Thus the above 30 ml eluatewas concentrated to remove methanol and diluted with 1M sodium citratepH 5.4 and methanol to 8 ml and a final solvent composition of 4:6methanol-0.1M sodium citrate. The solution was charged on 17 ml ofLiChroprep RP-18 resin. The column was eluted at 2 ml/min. successivelywith 75:25 methanol-0.1M sodium citrate pH 5.5 (Fractions 1-31, 2 mleach) and 8:2 methanol-citrate (Fractions 32-72, 2 ml each). Based onHPLC analysis Fractions 41-54 were combined as Rich Cut 1. The pH of therich cut was increased 1.5 units with dilute sodium hydroxide.Off-fractions 55-72 were combined, concentrated to a solvent compositionof 4:6 methanol-0.1M citrate and chromatographed as described above on17 ml of LiChroprep RP-18. Appropriate fractions were combined as RichCut 2. Solution pH was increased 1.5 units. Rich Cuts 1 and 2 werecombined, concentrated to remove methanol and adjusted to pH 7.2 with0.1M potassium phosphate pH 8.0. To the resulting 10 ml of cloudysolution was added sufficient methanol (7.5 ml) to clarify the sample.The solution was desalted by adsorption on 8 ml of LiChroprep RP-18resin. The resin was washed successively with 50 ml of 3:7 methanol-0.1Mpotassium phosphate pH 7.0 and 30 ml of 3:7 methanol-distilled water.The resin was then eluted with 30 ml of 9:1 methanol-water. The eluatecontained approximately 21 mg of substantially pure difficidin.

D. Isolation of Antibacterial Difficidin--Method 2

A 1.7 L aliquot of the 2.1 L spent ethyl acetate solution of Part Aabove was concentrated to an oil at 40° C. and diluted to 915 ml with9:1 methanol-water. Insoluble material was removed by centrifugation.The clear centrifugate was adsorbed at 20 ml/min on a column of 600 mlof DEAE-Sephadex A-25 (acetate cycle), packed in 9:1 methanol-water. Theresin was washed with 4 L of 9:1 methanol-water. Crude difficidincomponent was eluted from the resin with 3% ammonium chloride in 9:1methanol-water during which a series of 100 ml fractions were collected.The 450 ml rich cut, Fractions 7-10, contained 160 mg of difficidin byHPLC assay. The rich cut was desalted by diluting with 916 ml of waterand absorbing at 18 ml/min on 180 ml of Diaion HP-20 resin. The resinwas washed successively with 600 ml of water, 600 ml of 0.1M potassiumphosphate pH 7.5, and again 600 ml of water. Elution of the resin withmethanol afforded a 208 ml antibiotic rich cut containing 1 g of totalsolids. The sample was concentrated to 100 ml, diluted with 233 ml of0.05M potassium phosphate pH 7, and charged on a 190 ml column ofLiChroprep RP-18 resin, 25-40 micron. The column was then eluted at 10ml/min with 1:1 methanol-0.05M potassium phosphate pH7 (Fractions 1-40)followed by a linear gradient from 50% methanol to 90% methanol-buffer(Fractions 41-280). All fractions were 10 ml each. Based on HPLC assaysand in vitro agar disc diffusion assays against Vibrio percolans,Fractions 222-237 were pooled. The 154 ml rich cut contained 115 mg ofdifficidin. The sample was diluted with 170 ml of 0.05M potassiumphosphate pH 7 and adsorbed on a 95 ml Whatman Partisil M20 10/25 ODS-3column (2.2 cm 1D×25 cm), 10 micron. The column was eluted at 10 ml/minsuccessively with 65:35 methanol-0.05M potassium phosphate pH7(Fractions 1-89), 70:30 methanol-buffer (Fractions 90-149), and 75:25methanol-buffer (Fractions 150-230). All fractions were 5 ml each.Fractions 180-220 were combined and evaporated to 34 ml. Sufficientmethanol (11 ml) was added to clarify the resulting milky suspension.The solution was adsorbed at 2 ml/min on 18 ml of Diaion HP-20 resin.The resin was washed with 40 ml of 3:7 methanol-water. The resin waseluted with methanol to afford a rich cut containing 83 mg ofessentially pure difficidin, potassium salt. Lyophilization of theantibiotic from water gave a white amorphous solid, for which theanalytical results described below were obtained.

PHYSICOCHEMICAL CHARACTERIZATION OF DIFFICIDIN

soluble in water, lower alcohols such as methanol and ethanol;

pH of 0.1% by weight aqueous solution is 6.9;

    ______________________________________                                        UV Spectrum (in methanol)                                                            λ max                                                                        A.sup.1% .sub.1 cm                                               ______________________________________                                               235 nm                                                                              866                                                                     263 nm                                                                              316                                                                     273 nm                                                                              415                                                                     283 nm                                                                              328                                                              ______________________________________                                    

MASS SPECTRAL DATA

Upon trimethylsilylation, the sample exhibited a molecular ion at m/z688.3741 as a di-TMS derivative (M⁺.TMS₂) corresponding to the molecularformula C₃₁ H₄₅ O₆ P: MW 544 (calc'd for a di-TMS derivative, m/z688.3744) by high resolution mass measurement. Negative ion FAB (FastAtom Bombardment) analysis corroborated the molecular weight assignment(observed (M-H)⁻, m/z 543).

¹ H NMR SPECTRUM

The spectrum is set out as FIG. 1. The spectrum has been recorded at 300MHz in 20% CD₃ OD/CDCl₃ at 22° C. (˜5 mg/0.35 ml) and chemical shiftsare shown in ppm relative to internal tetramethylsilane at zero ppm.

¹³ C NMR CHEMICAL SHIFTS

The spectrum was recorded in 20% CD₃ OD/CDCl₃ at 5° C. Chemical shiftsare given in ppm downfield of internal tetramethylsilane (TMS) standard.In agreement with HRMS data, 31 carbon atoms are observed with thefollowing chemical shifts: 16.8, 18.7, 20.0, 23.2, 24.9, 30.7 (2X),32.7, 35.9, 39.0, 39.5, 41.5, 71.7, 74.2, 111.7, 115.4, 120.7, 123.6,124.2, 125.0, 126.1, 127.0, 127.2, 129.7, 133.2, 133.7, 134.6, 139.1,141.1, 146.2, 172.7 ppm.

EXAMPLE 3 Production of Difficidin and Oxydifficidin Using ATCC 39374Media Preparation

Medium A was prepared by suspending the ingredients listed below indistilled water. Fifty milliliters of this suspension was dispensed into250 ml three-baffled flasks and 500 ml into 2000 ml three-baffledflasks. The flasks were plugged with cotton and autoclaved 30 minutes at121° C., 18 PSI.

Medium B was prepared by suspending the ingredients listed below indistilled water. The pH was adjusted as indicated and 9.5 liters of thismedium was charged into a 14 L New Brunswick Scientific Microfermfermentor, model MF-114. The fermentor was autoclaved, unstirred, at121° C., 18 PSI, for 120 minutes. Following autoclaving, the medium wasmechanically agitated, aerated, and cooled to 28° C.

Medium C was prepared by suspending the ingredients listed below indistilled water, and heating to boiling, and dispensing 12 ml into18×150 mm tubes. Tubes were autoclaved twenty minutes at 121° C., 18PSI. Following autoclaving, the tubes were placed at an angle so thatthe medium would harden into slants.

Culture Preparation

A lyophilized tube of culture ATCC 39374 was inoculated aseptically intoa 250 ml three-baffled flask of Medium A and incubated at 220 RPM, 28°C., for 24 hours. This seed culture was used to inoculate 12 slants ofMedium C. Once inoculated, the slants were incubated for 96 hours at 28°C., then refrigerated. A portion of the slant prepared as indicatedabove, was used to inoculate a 250 ml flask of Medium A. The flask wasincubated at 220 RPM, 28° C., for 24 hours. Three percent of this firststage seed flask was used to inoculate a 2000 ml flask of medium A whichwas also incubated at 220 RPM, 28° C., for 24 hours. This second stageseed flask could then be used to either directly inoculate the fermentoror as the inoculum for a third stage seed. If a third stage seed wasused, three percent of the second stage seed was used to inoculateanother 2000 ml flask of Medium A. Following incubation of the thirdstage seed at 28° C., 220 RPM for 24 hours, the flask was used toinoculate the fermentor containing Medium B.

Production of Difficidin and Oxydifficidin

The fermentor containing Medium B prepared as described above andinoculated with the previously described second or third stage seedflask, was incubated at 28° C., 300-400 RPM, 0.3 vvm air. Sterilepolyglycol P-2000 was added to the fermentor as necessary to controlfoam; the total amount of polyglycol P-2000 in the fermentor did notexceed 1% (v/v). The fermentation was terminated at 88 hours. Sampleswithdrawn from the fermentor were extracted with methanol (1 part wholebroth plus 2 parts methanol) and assayed by HPLC for product formation.A sample profile, the average of two fermentations, is shown in TableVII. The harvested fermentors were stored at 4°-10° C. until processing.

    ______________________________________                                                        g/l                                                           ______________________________________                                        Medium A:                                                                     Trypticase Peptone                                                                              25                                                          Sucrose           10                                                          Soy Bean Flour    10                                                          Soluble Starch    10                                                          CaCO.sub.3        10                                                          Glucose           5                                                           Beef Extract      1                                                           Malt Extract      1                                                           Yeast Extract     1                                                           Corn Steep Liquor 0.5                                                         Soy Bean Oil      0.1                                                         Trypticase Soy    0.1                                                         pH 6.8 (as is)                                                                Medium B:                                                                     Dextrin           40                                                          Solulac           7                                                           Yeast Extract     5                                                           COCl.sub.2 --6H.sub.2 O                                                                         0.05                                                        Polyglycol P-2000 5 ml/L                                                      pH 7.3                                                                        Medium C:                                                                     Agar              20                                                          Malt Extract      10                                                          Dextrose          4                                                           Yeast Extract     4                                                           pH 7.0                                                                        ______________________________________                                    

                  TABLE VII                                                       ______________________________________                                        Activity Profile of 14-Liter Fermentations of                                 Difficidin and Oxydifficidin                                                  Age (Hours) Oxydifficidin (mg/l)                                                                        Difficidin (mg/l)                                   ______________________________________                                        16           9.2           12.2                                               28          25.6           61.1                                               40          27.2           61.8                                               53          41.4          116.4                                               64          46.5          132.7                                               88          55.4          149.2                                               ______________________________________                                    

These fermentation results are observed to be unusual in that the ratioof difficidin:oxydifficidin produced is about 3:1. More usually thisratio has been observed to be about 1:1.

EXAMPLE 4 Isolation of Difficidin

Approximately 11 L of whole broth (pH 6.5) from the fermentation batchdescribed in Example 3 above was harvested at 88 hours. Broth pH wasadjusted to 7.3 with aqueous sodium hydroxide. A 4.8 L portion ofisopropanol was added to the broth. After thorough mixing, the broth wasclarified by centrifugation. A 14.4 L sample of the clarified broth wasadsorbed at 100 ml/min on 1 L of Dowex 1×2 (Cl⁻) resin, 50-100 mesh. Theresin was washed with 2 L of distilled water and eluted at 100 ml/minwith 4 L of 3% ammonium chloride in 9:1 methanol-water. Eluate wascollected in eight 500 ml fractions. The 1.5 L rich cut (Fractions 2-4)was diluted with 3 L of water and adsorbed at 100 ml/min on a 1 L columnof Diaion HP-20 resin. The resin was washed successively with 2 L of 3:7methanol-water, 1.5 L of 0.1M potassium phosphate pH7, and 2 L of water.Elution of the resin with methanol afforded 2.5 g of 29% puredifficidin. The eluate was concentrated to 30 ml. A 10 ml aliquot (250mg difficidin) of the concentrate was diluted to 15.4 ml with 0.05Mpotassium phosphate pH 7.1 to bring the water concentration in thesample to 50% as determined by Karl Fischer analysis. The 15.4 ml samplewas chromatographed on 100 ml of Sephadex LH-20 resin in 1:1methanol-0.05M potassium phosphate pH 7.1. The flow rate was 2.5 ml/min.Difficidin eluted with a retention time of 1.75 column volumes. The 200ml rich cut was diluted with 150 ml of water and absorbed at 8 ml/min on80 ml of Diaion HP-20 resin equilibrated in 3:7 methanol-water. Theresin was washed with 100 ml of 3:7 methanol-water and eluted with 200ml of methanol. The eluate rich cut was 80 ml and contained 256 mg of56% pure difficidin. A 40 ml aliquot of the eluate was concentrated to 4ml, diluted to 8.2 ml with 0.03M potassium phosphate pH 7, and chargedon a 95 ml Whatman Partisil M20 10/25 ODS-3 column (10 micron) that hadbeen equilibrated in 65:35 methanol-0.03M phosphate buffer. The columnwas eluted with 75:25 methanol-buffer and effluent was collected in aseries of 10 ml fractions. Fractions 31 through 40 were combined,concentrated to a solution containing less than 30% methanol, andadsorbed at 2.5 ml/min on 25 mil of Diaion HP-20 resin. The resin waswashed with 100 ml of 3:7 methanol-water. Elution with methanol afforded54 mg of essentially pure difficidin. The antibacterial was stored inmethanol at -80° C. to minimize decomposition. Aliquots were lyophilizedfrom water to provide samples for physiochemical characterization. The'H NMR spectrum and mass spectra showed the product to be identical tothe product evaluated in Example 2, Step D above.

EXAMPLE 5 Preparation of Oxydifficidin

Three different fermentation procedures were employed in preparingoxydifficidin.

A. Media Preparation

Medium A, described below, was prepared by dissolving the ingredientslisted below in distilled water and adjusting the pH to between 7.0 and7.2 with concentrated HCl or 50% NaOH before sterilization. Fifty ml ofthis medium was dispensed into 250 ml, 3-baffled shake flasks, and 500ml of this same medium was dispensed into 2000 ml, 3-baffled shakeflasks. The flasks were plugged with cotton, sterilized by heating in anautoclave at 121° C. for 25 minutes, and allowed to cool to roomtemperature.

    ______________________________________                                        Medium A                                                                      ______________________________________                                        Dextrose               0.1%                                                   Soluble starch         1.0                                                    Beef extract           0.3                                                    Ardamine pH            0.5                                                    NZ Amine Type E        0.5                                                    MgSO.sub.4 7H.sub.2 O  0.005                                                  Phosphate Buffer.sup.1 2.0 ml                                                 CaCO.sub.3.sup. 2      0.05%                                                  ______________________________________                                         .sup.1 Phosphate Buffer:                                                      KH.sub.2 PO.sub.4 9.1%                                                        Na.sub.2 HPO.sub.4 9.5                                                        pH  7.70                                                                      .sup.2 Added after pH was adjusted to its appropriate level.             

Medium B, described below, was prepared in a New Brunswick Scientificnutrient sterilizer, model NS-20, by suspending the ingredients listedbelow in distilled water, adjusting the pH to between 7.2 and 7.4 withconcentrated HCl or 50% NaOH before sterilization, and sterilizing byheating at 121° C., 18 pounds per square inch of pressure, for 20minutes. Fourteen-liter New Brunswick Scientific magnetically coupledMicroferm fermentors, model MF-114, were sterilized with 100 mldistilled water in the jar by heating in an autoclave at 121° C., 18pounds per square inch of pressure, for 90 minutes. Nine and one-halfliters of cooled Medium B from the nutrient sterilizer was asepticallytransferred to the sterile fermentors and the temperature adjusted toabout 28° C.

    ______________________________________                                        Medium B                                                                      ______________________________________                                        Dextrin          4.0%                                                         Distillers Solubles                                                                            0.7                                                          Yeast Extract    0.5                                                          COCl.sub.2.6H.sub.2 O                                                                          0.005                                                        ______________________________________                                    

Culture Development

A lyophile of Bacillus subtilis ATCC 39320 was opened aseptically andused to inoculate a 250 ml flask of Medium A prepared as indicatedabove. The flask was incubated at 28° C. for 24 hours on a 220 rpmrotary shaker. Inoculum development continued by asepticallytransferring 15 ml from the first flask into each of three 2000 ml shakeflasks of Medium A. The 2000 ml shake flasks were incubated at 28° C.for 24 hours on a 220 rpm rotary shaker. Final pH measurements on theseed stages ranged from 7.20 to 8.35.

PRODUCTION

One 2000 ml flask was used to inoculate each of the 14-liter vesselsprepared as previously indicated. The fermentor conditions setimmediately after inoculation were 28° C. (27.9 to 28.3° C.), 300 rpm,and 2 liters/minute air, corresponding to a Kd_(w) of 2.35×10⁻⁴grams.moles O₂ /ml/hr/atm. Polyglycol P-2000 was added as necessaryduring the fermentation to control foam but did not exceed 0.04%. At 77hours post inoculation, the agitation and air were reduced to 200 rpmand 1 liter/minute, respectively, corresponding to a Kd_(w) of 1.3×10⁻⁴gram.moles O₂ /ml/hr/atm. Fermentors were harvested 89 hours postinoculation. Antibacterial and pH analyses were performed on fermentorfiltered broth samples and the data was as follows:

    ______________________________________                                        Age                 Antibiotic Activity.sup.1 vs                              (Hours)      pH     Vibrio percolans                                          ______________________________________                                         0           6.66    0                                                        17           7.20   26                                                        29           7.22   35                                                        41           7.08   34                                                        53           7.08   35                                                        65           6.65   28                                                        77           6.45   29                                                        89           6.35   29                                                        ______________________________________                                         .sup.l mm zones from 1/2" discs, 50 μl/disc.                          

B. Media preparation and culture development were the same as describedabove in A.

PRODUCTION

One 2000 ml flask was used to inoculate each of the 14 liter vesselsprepared as previously indicated. The fermentor conditions setimmediately after inoculation were 28° C. (28.0°-28.3° C.), 300 rpm, and2 liters/minute air, corresponding to a Kd_(w) of 2.35×10⁻⁴ grams.molesO₂ /ml/hr/atm. Polyglycol P-2000 was added as necessary during thefermentation to control foam but did not exceed 0.04%. Fermentors wereharvested 41 hours post inoculation. Antibacterial and pH analyses wereperformed on fementor filtered broth samples and the data was asfollows:

    ______________________________________                                        Age                 Antibiotic Activity.sup.1 vs                              (Hours)      pH     Vibrio percolans                                          ______________________________________                                         0           6.64    0                                                        17           6.40    0                                                        29           6.38   35                                                        41           6.45   32                                                        ______________________________________                                         .sup.1 mm zones from 1/2" discs, 50 μl/disc.                          

C. Media preparation was the same as described above in A.

CULTURE DEVELOPMENT

A lyophile of Bacillus subtilis ATCC 39320, was opened aseptically andused to inoculate a 250 ml flask of medium A prepared as indicatedabove. The flask was incubated at 28° C. for 24 hours on a 220 rpmrotary shaker. Inoculum development continued by asepticallytransferring 2 ml from the first flask to each of four other 250 mlflasks of Medium A. These four flasks were incubated at 28° C. for 24hours on a 220 rpm rotary shaker. After 24 hours, the four flasks werepooled and 15 ml was aseptically transferred to each of four 2000 mlshake flasks of Medium A. The 2000 ml shake flasks were incubated at 28°C. for 24 hours on a 220 rpm rotary shaker. Final pH measurements on theseed stages ranged from 7.21 to 8.35.

PRODUCTION

One 2000 ml flask was used to inoculate each of four 14 liter fermentorvessels prepared as previously indicated. The fermentation conditionsset immediately after inoculation were 28° C. (27.5°-28.2° C.), 400 rpm,and 3 liters/minute air corresponding to a Kd_(w) of 3.8×10⁻⁴grams.moles O₂ /ml/hr/atm. Polyglycol P-2000 was added as necessaryduring the fermentation to control foam but did not exceed 0.23%. At 65hours post inoculation, the agitation was lowered to 200 rpm, reducingthe Kd_(w) to 1.75×10⁻⁴ grams.moles O₂ /ml/hr/atm. Fermentors wereharvested 89 hours post inoculation. The four batches were combined andcentrifuged in a batch-fed Sharples centrifuge at 30,000 rpm. Thesupernatant was recentrifuged twice more, when a clear supernatant wasobtained. The cells were discarded and the supernatant retained forfurther processing. Antibacterial and pH analyses were performed onfermentor filtered broth samples and the data was as follows, averagedfor the four 14-liter batches:

    ______________________________________                                        Age                 Antibiotic Activity.sup.1 vs                              (Hours)      pH     Vibrio percolans                                          ______________________________________                                         0           6.93    5.8                                                      17           6.57   19.8                                                      29           6.38   27.7                                                      41           6.07   26.7                                                      53           6.11   26.7                                                      65           5.96   27.0                                                      77           6.50   27.5                                                      89           6.05   26.5                                                      ______________________________________                                         .sup.1 mm zones from 1/2" discs, 50 μl/disc.                          

EXAMPLE 6 Isolation and Purification of Oxydifficidin A. PreliminaryPurification of Oxydifficidin

Whole broth from four 14 L fermentations, described in Example 5 above,grown under identical conditions, were combined and processed through aturbo Sharples centrifuge. The clear centrifuge, 28.3 L, was adjusted topH 4.8 with concentrated hydrochloric acid and adsorbed on 2.1 L of Rohmand Haas Amberlite XAD-2 resin. The resin was then washed at the sameflow rate with 8 L of water. Antibiotic activity was eluted from theresin with 6 L of ethyl acetate, collected in a single fraction. Theelute contained approximately 0.8 L of a dark aqueous lower layer, whichwas removed and discarded. The ethyl acetate layer was dried brieflywith anhydrous sodium sulfate and then concentrated to 400 ml.

The crude antibacterial was back-extracted into water by stirring theethyl acetate concentrate with 150 ml of H₂ O during which the pH of theaqueous layer was brought to 8.5 with 2.5N sodium hydroxide. The aqueousextract was removed and the spent ethyl acetate was again extracted atpH 8.5 with 100 ml of water. After a final third extraction at pH 8.5with 70 ml of water, the three aqueous extracts were combined andadjusted to pH 8.5. Final volume 360 ml. The sample was stored frozen at-80° C.

B. Isolation of Essentially Pure Oxydifficidin

The combined aqueous extract from Step A above was thawed, concentratedto 205 ml, and buffered with 15 ml of 1M potassium phosphate pH 7. A 215ml aliquot of this solution was adsorbed on 500 ml of Dianion HP-20resin at a flow rate of 20 ml/min. The resin was stepwise washed with1.5 L of water, 1.5 L of 1:1 methanol-0.1M potassium phosphate pH 7, and1.5 L of 65:35 methanol-0.1M potassium phosphate pH 7. Oxydifficidin waseluted from the resin with 9:1 methanol-water. Effluent was collected in20 ml fractions and assayed by HPLC. Fractions 20 to 35 were combinedand concentrated to 50 ml (I). pH 7.5; 430 mg oxydifficidin by HPLCasay.

A 49 ml aliquot of (I) (421 mg oxydifficidin was charged on a 170 mlcolumn of LiChroprep RP-18 resin (E. Merck, 25-40 micron) in 1:4methanol-water. The column was first eluted with 250 ml of 1:4methanol-water at a flow rate of about 3.5 ml/min. Effluent wasdiscarded. The column was then eluted at the same flow rate with 1:1methanol-water. A series of 20 ml fractions were collected. Based on UVand HPLC assays, Fractions 27 to 38 were combined as essentially pureoxydifficidin. To prevent freezing during storage at -80° C., 100 ml ofmethanol was added to the fractions to bring the methanol concentrationto 65%. Final volume 360 ml containing 378 mg of essentially pureoxydifficidin.

The sample was stored at -80° C. (II).

C. Alkaline Phosphatase Dephosphonylation of Oxydifficidin

TLC System: Whatman KC₁₈ F reverse phase plate 9:1 methanol-0.1M sodiumcitrate pH 6.0.

To a 100 μl aliquot of a 5 mg/ml aqueous solution of oxydifficidin,sample (II) from Step B above, was added 10 μl of 0.25M TRIShydrochloride buffer pH 8.0 and 5 μl of calf intestinal mucosa alkalinephosphatase (2000 units/ml, P-L Biochemicals, Inc.). After 60 min.incubation at 28° C., the majority of oxydifficidin (R_(f) 0.59) hadbeen transformed into a new substance (R_(f) 0.38) which was negative toHane's Reagent for phosphate detection.

D. Isolation of Essentially Pure Oxydifficidin for Physicochemical andBiological Characterization

From fermentation studies with the oxydifficidin-producing organism,described in Example 5 above, three broths were obtained with goodantibiotic titer.

    ______________________________________                                        Batch        Whole Broth Volume                                                                           pH                                                ______________________________________                                        1            6.7 L          6.3                                               2              6 L          6.2                                               3            7.3 L          6.1                                               ______________________________________                                    

Crude oxydifficidin was isolated from each batch individually by thefollowing procedure. Whole broth (6-7 L) was adjusted to pH 4.8 withconcentrated hydrochloric acid. It was found advantageous to centrifugethe broth to remove the cell mass to avoid possible column pluggingduring the subsequent XAD-2 adsorption step. It was, however, shownpossible to adsorb oxydifficidin directly from whole broth onto XAD-2resin. The broth (clarified or whole) was adsorbed onto 700 ml of Rohmand Haas Amberlite XAD-2 resin, 20-50 mesh at 70 ml/min. The resin waswashed with 2.5 L of water. Crude oxydifficidin was eluted with 2.5 L ofethyl acetate. The lower aqueous layer of the ethyl acetate eluate wasdiscarded; the upper layer was dried over anhydrous sodium sulfate andconcentrated to ca. 300 ml. The ethyl acetate concentrated wasback-extracted at pH 8.5 successively with 100 ml, 50 ml, and 30 ml ofwater as described in Step B above. The combined aqueous extract wasadjusted to pH 6.8 and stored at -80° C.

The aqueous extracts of batches 1, 2, and 3 were each concentrated to150-200 ml to remove dissolved ethyl acetate and were then combined. Thesolution was buffered with 10 ml of 1M potassium phosphate pH 7 andadsorbed onto 450 ml of Dianion HP-20 resin at a flow rate of 20 ml/min.The column was washed successively with 1.4 L of 0.1M potassiumphosphate pH 7, 1.4 L of 1:1 methanol-0.1M potassium phosphate pH7. Thecolumn was then eluted with 9:1 methanol-water at 20 ml/min. Effluentwas collected in five fractions (Fr 1-5) with volumes of 220 ml, 100 ml,200 ml, and 210 ml respectively. Based on HPLC assay and agar discdiffusion assay against Vibrio percolans, Fractions 3 and 4 werecombined as the oxydifficidin cut (III); ca 350 mg oxydifficidin by HPLCassay.

Sample (III) was concentrated to 45 ml and adjusted to pH 7.0. Theconcentrate was charged on a column of 170 ml of LiChroprep RP-18 resin(E. Merck, 25-40 micron) in water. The column was first washed with 450ml of water. The column was then eluted with 600 ml of 1:1methanol-water, followed by 1.1 L of 65:35 methanol-water. The eluatewas collected in 20 ml fractions. Based on UV and HPLC assays, fractions28-42 were combined. To prevent freezing during storage at -80°, 100 mlof methanol was added to the pooled fractions (IV). Volume 380 ml. Thesample was lyophilized to afford 238 mg of essentially pureoxydifficidin (V). Stored under nitrogen at -80° C.

E. Physicochemical Characterization of Oxydifficidin (Sample V)

    ______________________________________                                        UV Spectrum (in 0.1 M potassium phosphate)                                           λMax                                                                           A.sup.1% .sub.1 cm                                             ______________________________________                                               235 nm  750                                                                   266 nm (sh)                                                                           304                                                                   275 nm  376                                                                   284 nm (sh)                                                                           307                                                            ______________________________________                                    

There is evidence that from 10-15% decomposition of oxydifficidin mayhave occurred during lyophilization of (IV) to (V). Based on thelyophilized weight of (V) and the UV analysis of (IV), A₁ cm^(1%) (275nm)--438 is calculated.

MASS SPECTRAL DATA

Electron impact mass spectra (EI-MS) gave M⁺ -H₃ PO₄ ions at m/z462.3095 corresponding to the formula C₃₁ H₄₂ O₃ (calc'd 462.3134) byhigh resolution mass measurements. Other significant ions in thehigh-mass region occur at m/z 444, 381 and 349. Upontrimethylsilylation, the sample exhibited a molecular ion at m/z776.4100 as a tri-TMS derivative (M⁺.TMS₃) corresponding to themolecular formula C₃₁ H₄₅ O₇ P (MW 560) calc'd for the tri-TMSderivative, m/z 776.4089). Other significant ions and theircorresponding exact mass values and elemental formula, confirming theneat EI-MS ions at m/z 462, 381 and 349 as mon-TMS derivatives, are asfollows:

    ______________________________________                                        Found        Calcd       Formula*                                             ______________________________________                                        534.3528     534.3529    C.sub.31 H.sub.42 O.sub.3.TMS.sub.1                  453.2881     453.2825    C.sub.25 H.sub.33 O.sub.3.TMS.sub.1                  421.2998     421.2927    C.sub.25 H.sub.33 O.TMS.sub.1                        299.0669     299.0720    H.sub.3 PO.sub.4.TMS.sub.3 --CH.sub.3                ______________________________________                                         *TMS = SiC.sub.3 H.sub.8                                                 

¹³ C NMR CHEMICAL SHIFTS

The spectrum was recorded in CD₃ OD/CDCl₃ (˜1:1) at 0° C. (12 mg/0.35ml). Chemical shifts are given in ppm downfield of internaltetramethylsilane (TMS) standard. In agreement with the mass spectraldata, 31 carbon atoms are observed with the following chemical shifts:16.3, 16.7, 18.8, 24.1, 26.1, 31.3 (2X), 33.5, 36.2, 41.8, 46.6, 72.5,74.5, 75.2, 114.1, 115.4, 121.3, 124.8, 125.0 (2X), 125.2, 126.6, 127.3,129.4, 133.9, 134.0, 137.1, 139.0, 141.2, 144.6, 173.5 ppm.

The peaks at 33.5 and 72.5 ppm occur as doublets through coupling to ³¹P of the phosphate group.

¹ H NMR SPECTRUM

The spectrum is set out as FIG. 2. The spectrum was recorded at 300 MHzin CD₃ OD/CDCl₃ (˜1:1) at 0° C. (˜4 mg/0.35 ml). Chemical shifts areshown in ppm relative to internal tetramethylsilane at zero ppm.

TLC Data

Whatman KC₁₈ F reverse phase plates

9:1 methanol-0.1M sodium citrate, pH 6.0 R_(f) =0.59

visualization methods

(1) illumination under short ultraviolet light

(2) Hane's reagent spray (ammonium molybdate-HClO₄) for phosphate esters

MS results establish the molecular formula for antibiotic oxydifficidinas C₃₁ H₄₅ O₇ P (MW 560). Facile loss of H₃ PO₄.2TMS from the molecularion of tris(trimethylsilyl)-oxydifficidin suggests the phosphorous atomis present as a phosphate monoester. This is supported by the positiveHane's reagent test (TLC) and by the ready inactivation of oxydifficidinby alkaline phosphatase Step C above. NMR data suggest that thestructure of oxydifficidin contains, in addition to the phosphate:

eight olefinic bonds, two of which are terminal methylenes

one ester or lactone function

one hydroxyl group, and

one ring.

What is claimed is:
 1. A compound of the formula: ##STR3## where R_(a)and R_(b) are members independently selected from the group consistingof hydrogen; alkali metal and alkaline earth metal cations; ammonium;and substituted ammonium; and R¹ is hydrogen or hydroxy.
 2. The compounddifficidin according to claim 1 wherein R¹ is hydrogen.
 3. The compoundoxydifficidin according to claim 1 wherein R¹ is hydroxy.
 4. Apharmaceutical composition for inhibiting the growth of bacteriacomprising an antibacterially effective amount of a compound accordingto claim 1 and a pharmaceutically acceptable carrier.
 5. A method oftreating bacterial infections in mammals which comprises intravenously,intramuscularly, or subcutaneously administering an antibacteriallyeffective amount of a compound according to claim 1.